Methods, reagents and kits for detecting minimal residual disease

ABSTRACT

The invention relates to the field of minimal residual disease (MRD) diagnostics, which is progressively more applied for the evaluation of treatment effectiveness in patients with a hematological malignancy, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL), B-cell chronic lymphocytic leukemia (B-CLL), and multiple myeloma (MM). Provided are unique reagent compositions with carefully selected and thoroughly tested combinations of antibodies, for ≥8-color flow cytometric stainings as well as for 10-color and 12-color flow cyometric stainings, which can reach sensitivities of at least 10−4, even down to 10−5. Also provided are diagnostic kits and methods for detecting MRD.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/812,384, filed Nov. 14, 2017 (pending), which is a divisional of U.S.patent application Ser. No. 14/407,268, filed Dec. 11, 2014 (abandoned),which is a 371 National Stage application of International ApplicationNo. PCT/NL2013/050420, filed Jun. 14, 2013, published in English, whichclaims the benefit of U.S. Provisional Application Ser. No. 61/659,524,filed on Jun. 14, 2012, each of which are incorporated herein byreference in their entirety.

The invention relates to the field of cancer diagnosis, morespecifically to means and method for the monitoring of diseasedevelopment during and after treatment or for the detection of minimaldisseminated disease. Cytostatic or cytotoxic treatment inducesremission in the majority of patients with lymphoid malignancies.Nevertheless many of these patients relapse. Apparently the currentcytostatic or cytotoxic treatment protocols are not capable of killingall malignant cells in these relapsing patients, although they reachedso-called complete remission according to cytomorphological criteria.Since the detection limit of cytomorphological techniques is not lowerthan 1-5% malignant cells, it is obvious that such techniques can onlyprovide superficial information about the effectiveness of treatment, upto 10¹⁰ tumor cells still potentially remaining in the body

Techniques with a higher sensitivity to detect “minimal residualdisease” or minimal disease (MRD) are needed to obtain better insight inthe reduction of tumor mass during induction treatment and furthereradication of the malignant cells during maintenance treatment from oneor more than one tissue. The application of flow cytometry for detectionof MRD is traditionally based on discrimination between malignant cellsand normal leukocytes via malignancy-associated phenotypiccharacteristics, such as aberrant expression, overexpression, andcross-lineage expression of antigens.

Current 4-color and 6-color flow cytometry reaches a fair sensitivity of10⁻³ (to 10⁻⁴) in most patients with a hematological malignancy.However, it should be noted that the detection of low frequencies ofmalignant cells in blood, bone marrow and other body fluids such ascerebrospinal fluid during and after therapy and after hematopoieticstem cell transplantation can be hampered by high frequencies of normalregenerating cells. The extent and the pattern of regeneration differsper treatment protocol, per phase of treatment, per time of sampling,and seems to be dependent on the intensity of the preceding treatment:the more intensive the treatment, the more prominent the regeneration ofhematopoietic cells.

Logically, both the background of regenerating cells and thedrug-induced immunophenotypic shifts reduce the sensitivity andspecificity of the existing 4-color and 6-color flow cytometric MRDmethods. This has mainly lead to approaches in which either multiplecombinations of markers are used to evaluate MRD in a patient or,alternatively to use one or a few patient-specific combinations ofmarkers.

Recognizing the need for improved diagnostic methods for MRD, thepresent inventors set out to identify additional markers which could beused to obtain a more sensitive and reliable assay for detecting MRD,particularly based on a fully integrated approach, in which informationof multiple markers is combined via multivariate analysis. In addition,this new approach is not anymore limited to individual patients, but isapplicable to every patient of a specific disease category, such B-cellprecursor acute lymphoblastic leukemia (BCP-ALL), B-cell chroniclymphocytic leukemia (B-CLL) and multiple myeloma (MM).

After careful selection of the relevant markers, design of appropriatecombinations of antibodies in multi-color tubes, and the selection ofsuited fluorochromes (based on need for brightness, compensation,stability, etc.), a set of antibody reagents was developed. The studieswere complemented with extensive multicentric evaluation of theconsensus panels in order to reshape and achieve an optimal efficiency.The inventors designed novel ≥8-color stainings with carefully selectedand thoroughly tested combinations of antibodies, which can reachsensitivities of 10⁻⁴ to 10⁻⁵ Based ondesign-testing-redesign-retesting-redesign (etc.), specific combinationsof fluorochrome-conjugated antibodies have been developed per diseasecategory, such as BCP-ALL, B-CLL and MM. One or two ≥8-colorcombinations per patient will allow careful MRD monitoring withsensitivities of at least 10⁻⁴. The provided 10-color and 12-colorantibody combinations can even better discriminate between normal cellsand their malignant counterparts, thereby allowing for MRD detectionwith sensitivities down to 10⁻⁵.

Here we present novel 8-color 10-color and 12-color antibodycombinations for detection of MRD in a sample, e.g. blood or bonemarrow, isolated from patients with:

-   -   B-cell precursor acute lymphoblastic leukemia (BCP-ALL)    -   B-cell chronic lymphocytic leukemia (B-CLL) or    -   Multiple myeloma (MM) and related plasma cell disorders (PCD).

These multi-color immunostainings can be performed according to theso-called EuroFlow protocols as described by Van Dongen et al. Leukemia2012; 26: 1908-1075 and by Kalina et al. Leukemia 2012; 26: 1986-2010.

Accordingly, the invention provides unique reagent compositions for flowcytometric detection of MRD, comprising a combination of at least eightdistinct fluorochrome-conjugated antibodies. In particular, the reagentcompositions are of use for detecting MRD in patients with BCP-ALL,B-CLL or MM/PCD. In a preferred embodiment, the composition comprisesmonoclonal antibodies against a given CD antigen. CD stands for clusterdesignation and is a nomenclature for the identification of specificcell surface antigens or intracellular antigens defined by monoclonalantibodies. (Monoclonal) antibodies against the indicated markers can becommercially obtained from various companies, including Becton/Dickinson(BD) Biosciences, Dako, Beckman Coulter, CYTOGNOS, Caltag, Pharmingen,Exbio, Sanquin, Invitrogen, and the like.

Flow Cytometric MRD Detection in BCP-ALL

In one embodiment, the invention provides a reagent composition for flowcytometric detection of BCP-ALL cells in a human subject, comprising apanel of at least eight distinct fluorochrome-conjugated antibodies. TheBCP-ALL panel comprises antibodies against the four “core markers” CD10,CD19, CD20, CD34 and CD45.

Preferably, the panel further comprises one or more antibodies selectedfrom the group of antibodies against CD38, CD81, CyIgμ, anddeoxynucleotidyl transferase (NuTdT). Very good results are obtained ifthe panel further comprises one or more sets of antibodies selected from(a) set of antibodies against CD66c and CD123; (b) set of antibodiesagainst CD304 and CD73; and (c) set of antibodies against SmIgκ andSmIgλ, wherein the antibodies within each set are conjugated to the samefluorochrome. In a specific aspect, the BCP-ALL panel comprisesantibodies against CD10, CD19, CD20, CD34, CD45, one or more antibodiesselected from the group of antibodies against CD38, CD81, CyIgμ, NuTdT,and two or more sets of antibodies selected from (a) set of antibodiesagainst CD66c and CD123; (b) set of antibodies against CD304 and CD73;and (c) set of antibodies against SmIgκ and SmIgλ, wherein theantibodies within each set are conjugated to the same fluorochrome.

For example, a reagent composition comprises distinctfluorochrome-conjugated antibodies directed against one of the followingcombinations of markers:

-   -   (i) CD20, CD45, CD81, CD66c, CD123, CD34, CD19, CD10 and CD38,        wherein the antibodies against CD66c and CD123 are conjugated to        the same fluorochrome;    -   (ii) CD20, CD45, CD81, CD304, CD73, CD34, CD19, CD10 and CD38,        wherein the antibodies against CD304 and CD73 are conjugated to        the same fluorochrome;    -   (iii) CD20, CD45, NuTdT, SmIgκ, SmIgλ, CyIgμ, CD19, CD34 and        CD10, wherein the antibodies against SmIgκ and SmIgλ are        conjugated to the same fluorochrome. See for instance the        8-color BCP-ALL MRD Panel 1A.

As another example, it comprises distinct fluorochrome-conjugatedantibodies directed against the markers CD20, CD45, CD81, NuTdT, CD34,CD19, CD10 and CD38, and one or more sets of antibodies selected from(a) set of antibodies against CD66c and CD123; (b) set of antibodiesagainst CD304 and CD73; and (c) set of antibodies against SmIgκ andSmIgλ, wherein the antibodies within each set are conjugated to the samefluorochrome. See for instance the 10-color tube in Panel 1B comprisingantibodies against the markers CD20, CD45, CD81, NuTdT, CD66c, CD123,CD304, CD73, CD34, CD19, CD10 and CD38.

In a further specific aspect, the composition comprises a combination offluorochrome-conjugated antibodies directed against the markers CD20,CD45, CD81, NuTdT, CD66c, CD123, CD304, CD73, SmIgκ, SmIgλ, CyIgμ, CD34,CD19, CD10 and CD38, wherein the antibodies against each of the setsCD66c/CD123, CD304/CD73 and SmIgκ/SmIgλ are conjugated to the samefluorochrome. See for instance the 12-color tube in panel 1C.

Suitable fluorochromes for conjugating antibodies for use in the presentinvention against the recited markers are known in the art. As will beunderstood, the fluorochromes used within a reagent composition shouldbe distinguishable from each other by flow cytometry. The fluorochromesare preferably selected for brightness, limited spectral overlap andlimited need for compensation, stability, etc (see: Kalina et al.Leukemia 2012: 26: 1986-2010).

The following panel of fluorochromes is of particular use in a BCP-ALLreagent composition according to the invention: (1) pacific blue (PacB),brilliant violet 421 (BV421) or Horizon V450, (2) pacific orange (PacO),Horizon V500 (HV500), BV510, Khrome orange (KO) or 00515, (3)fluorescein isothiocyanate (FITC) or Alexa488, (4) phycoerythrin (PE),(5) peridinin chlorophyl protein/cyanine 5.5 (PerCP-Cy5.5), PerCP orPE-TexasRed, (6) phycoerythrin/cyanine7 (PE-Cy7), (7) allophycocyanine(APC) or Alexa647, and (8) allophycocyanine/hilite 7 (APC-H7), APC-Cy7,Alexa680, APC-A750, APC-C750 or Alexa700. After multiple testing rounds,the present inventors observed that very good results can be obtained ifthe following fluorochromes are chosen: Pacific Blue, brilliant violet421 or Horizon V450, PacO or Horizon V500, FITC, PE, PerCP-Cy5.5,PE-Cy7, APC, and APC-H7 or APC-A750 or APC-C750. In a specific aspect,the invention provides for a reagent composition shown in Table 1, panel1A, panel 1B or panel 1C.

TABLE 1 Exemplary reagent compositions for MRD detection in BCP-ALL..Panel 1A. Marker Composition of 8-color BCP-ALL MRD panels of theinvention tube PacB PacO FITC PE PerCPCy5.5 PECy7 APC APCC750 1 CD20CD45 CD81 CD66c and CD123 CD34 CD19 CD10 CD38 2 CD20 CD45 CD81 CD304 andCD73 CD34 CD19 CD10 CD38 2 CD20 CD45 NuTdT SmIgκ and SmIgλ CyIgμ CD19CD34 CD10 Panel 1B. Marker Composition of 10-color BCP-ALL MRD panel ofthe invention F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 CD20 CD45 CD81 NuTdT CD66cand CD123 CD304 and CD73 CD34 CD19 CD10 CD38 Panel 1C. MarkerComposition of 12-color BCP-ALL MRD panel of the invention F1 F2 F3 F4F5 F6 F7 F8 F9 F10 F11 F12 CD20 CD45 CD81 NuTdT CD66c and CD123 CD304and CD73 SmIgκ and SmIgλ CyIgμ CD34 CD19 CD10 CD38

Flow Cytometric MRD Detection in B-CLL

In another embodiment, the invention provides a reagent composition forflow cytometric detection of B-cell chronic lymphocytic leukemia (B-CLL)in a human subject, comprising a panel of at least eight distinctfluorochrome-conjugated antibodies, the panel comprising at leastantibodies against the seven “core markers” CD5, CD27, CD79b, CD3,CD200, CD81 and CD19. It was found that CD22 and/or Receptor tyrosinekinase-like orphan receptor 1 (ROR1) can be used as valuable additionalmarker(s). Very good results were obtained in combination with themarkers CD43 and CD38.

Preferred marker combinations for detecting B-CLL are as follows:

(a) CD27, CD3, CD79b, CD5, CD22, CD19, CD200 and CD81 (b) CD5, CD3,CD79b, ROR1, CD27, CD19, CD200 and CD81 (c) CD27, CD3, CD79b, ROR1, CD5,CD22, CD19, CD20, CD200 and CD81 (d) CD27, CD3, CD79b, ROR1, CD5, CD22,CD19, CD20, CD200, CD43, CD81 and CD38.

The following panel of fluorochromes is of particular use in a CLLreagent composition according to the invention: (1) pacific blue (PacB),brilliant violet 421 (BV421) or Horizon V450, (2) pacific orange (PacO),Horizon V500 (HV500), BV510, Khrome orange (KO) or 00515, (3)fluorescein isothiocyanate (FITC) or Alexa488, (4) phycoerythrin (PE),(5) peridinin chlorophyl protein/cyanine 5.5 (PerCP-Cy5.5), PerCP orPE-TexasRed, (6) phycoerythrin/cyanine7 (PE-Cy7), (7) allophycocyanine(APC) or Alexa647, and (8) allophycocyanine/hilite 7 (APC-H7), APC-Cy7,Alexa680, APC-A750, APC-C750 or Alexa700. After multiple testing rounds,the present inventors observed that very good results can be obtained ifthe following fluorochromes are chosen: Pacific Blue, brilliant violet421 or Horizon V450, PacO or Horizon V500, FITC, PE, PerCP-Cy5.5,PE-Cy7, APC, and APC-H7 or APC-A750 or APC-C750.

In a specific aspect, the invention provides for a reagent compositionshown in Table 2.

TABLE 2 Exemplary reagent compositions for MRD detection in B-CLL Panel2A. Composition of 8-color CLL MRD panel BV421 BV510 FITC PE PerCPCy5.5PECy7 APC APCC750 CD27 CD3 CD79b CD5 CD22 CD19 CD200 CD81 CD5 CD3 CD79bROR1 CD27 CD19 CD200 CD81 Panel 2B. Compositin of 10-color CLL MRD tubeF1 F2 F3 F4 F5 F6 F7 F8 F9 F10 CD27 CD3 CD79b ROR1 CD5 CD22 CD19 CD20CD200 CD81 Panel 2C. Composition of 12-color CLL MRD tube F1 F2 F3 F4 F5F6 F7 F8 F9 F10 F11 F12 CD27 CD3 CD79b ROR1 CD5 CD22 CD19 CD20 CD200CD43 CD81 CD38

Flow Cytometric MRD Detection in Multiple Myeloma/Plasma Cell Disease(MM/PCD)

A still further aspect of the invention relates to a reagent compositionfor detecting MM or PCD cells. The panel comprises antibodies againstthe four “core markers” CD138, CD38, CD56 and CD19, supplemented with atleast four additional markers selected from the group consisting ofCD27, CD117, CD81, CD229, CD45, CyIgκ and CyIgλ, CD45 is a preferredfifth marker, preferably in combination with CD27, CD117 and CD81 orCD229, CyIgκ and CyIλ.

Provided is a reagent composition with distinct fluorochrome-conjugatedantibodies against either one of the following panels of markers:

(n) CD45 CD CD56 CD27 CD19 CD117CD81 (o) CD45 CD138CD38 CD56 CD229CD19CyIgκ CyIgλ (p) CD138 CD27 CD38 CD56 CD45 CD19 CD117CD81 (q) CD138 CD27CD38 CD56 CD229CD19 CyIgκ CyIgλ (r) CD138 CD 27 CD38 CD56 CD45 CD19CyIgκ CyIgλ

For example, provided is a reagent composition for flow cytometricdetection of MM or PCD in a human subject, comprising a panel of atleast eight distinct fluorochrome-conjugated antibodies, the panelcomprising at least antibodies against the core markers CD138, CD38,CD56 and CD19, supplemented with at least four additional markersselected from the group consisting of CD27, CD117, CD81, CD229, CD45,CyIgκ and CyIgλ. Preferably, CD45 is the fifth marker, more preferablyin combination with the markers CD27, CD117 and CD81, or in combinationwith the markers CD229, CyIgκ and CyIgλ. Preferred reagent compositionscomprise distinct fluorochrome-conjugated antibodies directed againstone of the following combinations of markers:

(iv) CD45, CD138, CD38, CD56, CD27, CD19, CD117 and CD81 (v) CD45,CD138, CD38, CD56, CD229, CD19, CyIgκ and CyIgλ (vi) CD138, CD27, CD38,CD56, CD45, CD19, CD117 and CD81

(vii) CD138, CD27, CD38, CD56, CD229, CD19, CyIgκ and CyIgλ(viii) CD138, CD27, CD38, CD56, CD45, CD19, CyIgκ and CyIgλ See forinstance the 8-color PCD MRD Panel 3A.

Very good results were obtained using fluorochrome-conjugated antibodiesdirected against the markers CD138, CD27, CD38, CD56, CD45, CD19, CD117,CD81 and one or both set(s) of antibodies selected from (a) set ofantibodies against CD229 and CD28; and (b) set of antibodies againstCyIgκ and CyIλ. See for instance the 10-color tube in Panel 3B and the12-color tube in Panel 3C.

The following panel of fluorochromes is of particular use in a MM/PCDreagent composition according to the invention: (1) pacific blue (PacB),brilliant violet 421 (BV421) or Horizon V450, (2) pacific orange (PacO),Horizon V500 (HV500), BV510, Khrome orange (KO) or 00515, (3)fluorescein isothiocyanate (FITC) or Alexa488, (4) phycoerythrin (PE),(5) peridinin chlorophyl protein/cyanine 5.5 (PerCP-Cy5.5), PerCP orPE-TexasRed, (6) phycoerythrin/cyanine7 (PE-Cy7), (7) allophycocyanine(APC) or Alexa647, and (8) allophycocyanine/hilite 7 (APC-H7), APC-Cy7,Alexa680, APC-A750, APC-C750 or Alexa700. After multiple testing rounds,the present inventors observed that very good results can be obtained ifthe following fluorochromes are chosen: Pacific Blue, brilliant violet421 or Horizon V450, PacO or Horizon V500, FITC, PE, PerCP-Cy5.5,PE-Cy7, APC, and APC-H7 or APC-A750 or APC-C750.

TABLE 3 Exemplary reagent compositions for MRD detection in MM/PCD Panel3A. Composition of PCD MRD panel PacB or APCH7 or BV421 or HV500 orAPCA750 or Tube HV450 PacO FITC PE PerCPCy5.5 PECy7 APC APCC750 1 CD45CD138 CD38 CD56 CD27 CD19 CD117 CD81 2 CD45 CD138 CD38 CD56 CD229 CD19CyIgκ CyIgλ 3 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD81 4 CD138 CD27CD38 CD56 CD229 CD19 CyIgκ CyIgλ 5 CD138 CD27 CD38 CD56 CD45 CD19 CyIgκCyIgλ Panel 3B. Composition of 10-color PCD MRD panel F1 F2 F3 F4 F5 F6F7 F8 F9 F10 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD81 CD229 CD28 CD138CD27 CD38 CD56 CD45 CD19 CD117 CD81 CyIgκ CyIgλ Panel 3C. Composition of12-color MCD MRD tube F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 CD138 CD27CD38 CD56 CD45 CD19 CD117 CD81 CD229 CD28 CyIgκ CyIgλ

A further aspect of the invention relates to a diagnostic kit fordetecting MRD, in particular BCP-ALL MRD, CLL MRD or MM/PCD MRDcomprising one or more of the reagent compositions described hereinabove, optionally together with instructions for use, buffer, and/orcontrol samples (see: Kalina et al. Leukemia 2012: 26: 1986-2010). Inone embodiment, there is provided a BCP-ALL kit comprising one or morereagent compositions of Table 1. In another embodiment, there isprovided a CLL kit comprising one or more reagent compositions of Table2. In still a further embodiment, there is provided a PCD kit comprisingone or more reagent compositions of Table 3.

The invention also relates to a method for flow cytometric detection ofMRD, comprising the steps of providing a biological sample from a humansubject and contacting at least a portion (aliquot) of the sample with areagent composition provided herein. Any type of sample known orsuspected to contain leukocytes may be used directly, or after lysingnon-nucleated red cells, or after density centrifugation, or after cellsorting procedures. For example, the sample is peripheral blood, bonemarrow, tissue sample such as lymph nodes, adenoid, spleen, or liver, orother type of body fluid such as cerebrospinal fluid, vitreous fluid,synovial fluid, pleural effusions or ascitis. Peripheral blood or bonemarrow is preferred.

Provided is a multi-color flow cytometric method for detecting minimalresidual disease (MRD) in a biological sample comprising cells,preferably B-lineage cells (B-cell precursors, B-lymphocytes, and plasmacells), comprising the steps of:

(i) staining the sample with a reagent composition according to theinvention,(ii) subjecting the sample to flow cytometry; (iii) gating on cells forexpression of the selected markers detected by the antibodies present inthe reagent composition;(iv) distinguishing between normal and malignant cells, based on theexpression profile of the multiple markers.

Preferably, the analysis in step (iv) involves multivariate analysis,preferably principal component analysis (PCA), wherein each marker hasadded value in the distinction process via the principal componentanalysis. Advantageously, using automated population separation—APSview—is used employing for example Infinicyt software, ormultidimensional scaling (MDS) analysis.

PCA is a mathematical procedure that uses an orthogonal transformationto convert a set of observations of possibly correlated variables into aset of values of uncorrelated variables called principal components. Thenumber of principal components is less than or equal to the number oforiginal variables. This transformation is defined in such a way thatthe first principal component has as high a variance as possible (thatis, accounts for as much of the variability in the data as possible),and each succeeding component in turn has the highest variance possibleunder the constraint that it be orthogonal to (uncorrelated with) thepreceding components. Principal components are guaranteed to beindependent only if the data set is jointly normally distributed. PCA issensitive to the relative scaling of the original variables. Dependingon the field of application, it is also named the discreteKarhunen-Loève transform (KLT), the Hotelling transform or properorthogonal decomposition (POD). Alternatively to PCA, MDS or any othertype of well-established multivariate analysis, can be used (see:Pedreira et al. Trends Biotechnol 2013).

In one embodiment, there is provided is a multi-color flow cytometricmethod for detecting minimal residual disease (MRD) in a biologicalsample comprising lymphocytes, wherein MRD is BCP-ALL, comprising thesteps of:

(i) staining the sample with a BCP-ALL reagent composition according tothe invention, preferably a composition selected from any one of panels1A, 1B or 1C;(ii) subjecting the sample to flow cytometry; (iii) gating on mature Bcells and BCP-cells for expression of the markers detected by theantibodies present in the reagent composition; (iv) distinction betweennormal and malignant BCP cells, based on the application of multiplemarkers, each having added value in the distinction process via theprincipal component analysis. See examples in Example 1 and FIGS. 1 and2 for an exemplary analysis allowing for detection of MRD in BCP-ALLpatients.

In another embodiment, there is provided is a multi-color flowcytometric method for detecting minimal residual disease (MRD) in abiological sample comprising lymphocytes, wherein MRD is CLL, comprisingthe steps of:

(i) staining the sample with a CLL reagent composition according to theinvention, preferably a composition selected from any one of panels 2A,2B or 2C;(ii) subjecting the sample to flow cytometry; (iii) gating B-lymphocytesfor expression of the markers detected by the antibodies present in thereagent composition; (iv) distinction between normal and malignant Bcells, based on the application of multiple markers, each having addedvalue in the distinction process via the principal component analysis.

See examples in Example 2 and FIGS. 3 and 4 for an exemplary analysisallowing for detection of MRD in CLL patients.

In yet another embodiment, there is provided is a multi-color flowcytometric method for detecting minimal residual disease (MRD) in abiological sample comprising lymphocytes, wherein MRD is MM/PCD,comprising the steps of:

(i) staining the sample with a MM/PCD reagent composition according tothe invention, preferably a composition selected from any one of panels3A, 3B or 3C;(ii) subjecting the sample to flow cytometry; (iii) gating plasma cellsfor expression of the markers detected by the antibodies present in thereagent composition; (iv) distinction between normal and malignantplasma cells, based on the application of multiple markers, each havingadded value in the distinction process via the principal componentanalysis.See examples in Example 3 and FIGS. 5 and 6 for an exemplary analysisallowing for detection of MRD in MM/PCD patients.

LEGEND TO THE FIGURES

FIG. 1. Typical example of how to use the CD19 and CD45 identificationmarkers in combination with SSC (Panel A to C) for the distinctionbetween BCP cells and other nucleated cells in a bone marrow sample froma BCP-ALL patient during therapy. In each plot, light grey eventscorrespond to non-B cells in the sample, while dark grey events aremature B-cells and black events BCP cells. In the multivariate analysisrepresentation (APS1) of principal component 1 vs. principal component 2(Panel D), both mature B cells and BCP cells are clearly separated fromall other events based on all informative parameters (e.g. CD19, CD45,SSC).

FIG. 2.—Illustrating example of how to use immunophenotypiccharacterization markers CD10, CD20, CD34, CD66c/CD123, and CD38 incombination with SSC for the distinction between BCP-ALL cells andnormal residual B-cells in a bone marrow sample from a BCP-ALL patientduring therapy (Panels A to D). Only bone marrow B-cells are displayed,after gated/selected as described in FIG. 1. In each plot, black dotscorrespond to BCP-ALL cells in the sample, while grey dots are normalB-cells. Compared to normal B-cells, BCP-ALL cells show overexpressionof CD81 (Panel D), CD10 (Panels A and D), and CD66c/CD123 (Panel C). Inthe APS1 (principal component 1 versus principal component 2)representation based on all immunophenotypic markers and scattercharacteristics evaluated (Panel E), normal residual B-cells (grey) areclearly discriminated from BCP-ALL cells (black).

FIG. 3.—Illustrating example of how to use the CD19 and CD3identification markers in combination with SSC (Panels A to C) for thedistinction between mature B-cells and other nucleated cells in aperipheral blood sample from a CLL patient. In each plot, grey eventscorrespond to non-B-cells in the sample, while black events are totalperipheral blood B-cells. In the multivariate analysis representation(APS1) of principal component 1 vs. principal component 2 (Panel D),B-cells are clearly separated from all other events based on allinformative parameters (e.g. CD19, CD3, SSC).

FIG. 4.—Illustrating example of how to use immunophenotypiccharacterization markers CD27, CD5, CD22, CD200 and CD79b (panels A toC) for the distinction between CLL cells and normal mature B-cells in aperipheral blood sample from a CLL patient. Only peripheral bloodB-cells are displayed, after gated/selected as described in FIG. 3. Ineach plot, black dots correspond to CLL cells in the sample, while greydots are normal peripheral blood B-cells. Compared to normal B-cells,CLL cells show underexpression of CD22 (Panel B) and CD79b (Panel C)together with overexpression of CD200 (Panel B) and CD5 (Panels A andC). In the APS1 (principal component 1 vs. principal component 2)representation based on all immunophenotypic markers and scattercharacteristics evaluated (Panel D), CLL cells are clearly discriminatedfrom normal B-cells, while this degree of discrimination could not beachieved based on individual markers.

FIG. 5.—Illustrating example of how to use the CD38 and CD138identification markers in combination with SSC (Panel A to C) for thedistinction between plasma cells and other nucleated cells in a bonemarrow sample of an MM patient after therapy. In each plot, grey dotscorrespond to non-plasma cells in the sample, while black dots are totalbone marrow plasma cells. In the multivariate analysis representation(APS1) of principal component 1 vs. principal component 2 (Panel D),plasma cells (black dots) are clearly separated from all other events(grey dots) based on all informative parameters (e.g. CD138, CD38, SSC).

FIG. 6.—Illustrating example of how to use immunophenotypiccharacterization markers CD81, CD19, CD45, CD56, CD27, CD117 and CD38,in combination with SSC (Panels A to D) for the distinction betweenmyeloma/malignant plasma cells and normal residual plasma cells in abone marrow sample of an MM patient after therapy. Only bone marrowplasma cells are displayed, after gated/selected as described in FIG. 5.In each plot, black dots correspond to myeloma/clonal plasma cells inthe sample, while grey dots are normal residual bone marrow plasmacells. Compared to normal plasma cells, myeloma/clonal plasma cells showunderexpression of CD81, CD19, CD45, CD27 and CD38 together with higherSSC and overexpression of CD56 and CD117. In the APS1 (principalcomponent 1 vs. principal component 2) representation (Panel E) based onall immunophenotypic markers and scatter characteristics evaluatednormal residual plasma cells (grey dots) are clearly discriminated frommyeloma/malignant plasma cells (black dots), while this degree ofdiscrimination could not be achieved based on individual markers.

EXPERIMENTAL SECTION

The power of the EuroFlow approach disclosed herein is based on thecombination of sets of markers and the usage of multivariate analysesfor both the identification of normal cells (e.g. normal precursorB-cells, normal B-lymphocytes and normal plasma cells) and thedistinction between normal/reactive cells vs. clonal/malignant cells.For this purpose, a powerful multivariate analysis of the contributionof individual markers for inclusion and exclusion of each marker in thepanel according to its contribution over all other markers in thecombination. Such a strategy was used to evaluate the selectedcombinations of most discriminating markers in multiple sequentialrounds of experimental testing. Because of this the final proposedantibody combinations became extremely strong when used in combinationwith the principal component analysis, specifically with the automatedpopulation separation (APS) tool of the Infinicyt software, so that theadded (independent) value of each marker is used in a single step ofanalysis.

Herewith we provided the summary of the results of the extensiveexperimental studies for MRD detection in blood and bone marrow ofpatients with BCP-ALL (Example 1), CLL (Example 2) and Multiple Myeloma(Example 3).

In the Examples below, lists of markers are provided together with themost frequent phenotypic aberration of these markers in case of BCP-ALL,CLL, and MM/PCD. However, it should be noted that the realdiscrimination power between normal and malignant cells is based oncombinations of markers in the corresponding n-dimensional space, as isclearly visible in the principle component analyses in the figures ofExamples 1 to 3. In fact, minor differences of several markers add up toa larger difference in principle component analysis. Therefore thecurrent invention is not about single marker studies for MRD detection,but about carefully selected sets of markers, that allow excellentdiscrimination between normal cells and their malignant counterparts,such as normal BCP cells vs. BCP-ALL blasts, normal B-lymphocytes vs.B-CLL cells, and normal plasma cells vs. MM/PCD plasma cells.

Example 1. Antibody Panels and Diagnostic Method for MRD Detection inBCP-ALL Patients Markers for Identification of Total B Cells and B-CellPrecursors in the Bone Marrow

List of relevant identification markers: CD19, CD45

How to use them: Pre-gating using the CD19 marker is essential foridentifying a pure B-cell population. To focus on normal B-cellprecursors (BCP), CD45-negativity or weak positivity can be used todiscriminate BCP from CD45-positive mature B-cells. In case ofCD19-directed therapies, CD19 might be replaced by CD22. These markersmay be used in combination also with sideward light scatter (SSC) orforward light scatter (FSC) or both FSC and SSC to identify B-cells inperipheral blood or bone marrow or other types of samples (e.g. bonemarrow, tissue biopsy, spinal fluid). Of note, other markers, like CD10,CD20, CD38 and CD34, which are used for discriminating BCP-ALL cellsfrom normal BCP cells (see below), may also contribute to the gating ofthe total BCP cell population (e.g. CD34+, CD10+, CD20-to dim, CD38+).

Markers for Distinguishing Normal Vs. Malignant B-Cell Precursor Cells

List of markers and most frequent phenotypic aberration:

-   -   CD38: underexpressed in BCP-ALL/malignant vs. normal B-cell        precursor cells    -   CD10: over- or underexpressed in BCP-ALL/malignant B-cell        precursor cells    -   CD45: underexpressed (usually negative) in BCP-ALL/malignant vs.        normal B-cell precursor cells    -   CD20: under- or overexpressed in BCP-ALL/malignant vs. normal        B-cell precursor cells    -   CD81: over- or underexpressed in BCP-ALL/malignant vs. normal        B-cell precursor cells    -   CD66c: overexpressed in BCP-ALL/malignant vs. normal B-cell        precursor cells (particularly BCR-ABL positive ALL; generally        negative in TEL-AML1-positive or MLL-AF4-positive ALL)    -   CD123: overexpressed in BCP-ALL/malignant vs. normal B-cell        precursor cells (particularly in hyperdiploid ALL)    -   CD304: overexpressed in BCP-ALL/malignant vs. normal B-cell        precursor cells    -   CD73: overexpressed in BCP-ALL/malignant vs. normal B-cell        precursor cells    -   CD34: under- or overexpressed in BCP-ALL/malignant vs. normal        B-cell precursor cells    -   SSC: increased or decreased intensity in BCP-ALL/malignant vs.        normal B-cell precursor cells.    -   FSC: increased or decreased intensity in BCP-ALL/malignant vs.        normal B-cell precursor cells.

Example 2. Antibody Panels and Diagnostic Method for MRD Detection inCLL Patients Markers for Identification of Total B-Cells in PeripheralBlood and Bone Marrow:

List of identification markers: CD19, CD3 (exclusion marker)

How to use them: Pre-gating using this marker combination is essentialfor identifying a pure B-cell population, and removing T-cell/B-celldoublets. These markers may be used in combination also with sidewardlight scatter (SSC) or forward light scatter (FSC) or both FSC and SSCto identify B-cells in peripheral blood or bone marrow or other types ofsamples (e.g. tissue biopsy, spinal fluid). For a more refined gatingwith better enrichment of CLL cells, both CD5 and CD27 may be used.

Markers for Distinguishing Normal B-Cells from CLL Cells:

List of markers and most frequent phenotypic aberration:

-   -   CD27: positive on CLL cells and a small fraction of normal        B-cells    -   CD5: positive on CLL cells and a small fraction of normal        B-cells    -   CD79b: underexpressed on CLL cells as compared to normal        transitional and mature B-lymphocytes    -   CD22: underexpressed on CLL cells as compared to normal        transitional and mature B-lymphocytes    -   CD20: underexpressed on CLL cells as compared to normal        transitional and mature B-lymphocytes    -   CD200: overexpressed on CLL cells as compared to normal        transitional and mature B-lymphocytes

ROR1: overexpressed on CLL cells as compared to normal transitional andmature B-lymphocytes

-   -   CD43: overexpressed on CLL cells as compared to normal        transitional and mature B-lymphocytes    -   CD81: underexpressed on CLL cells as compared to B-cell        precursors and both transitional and mature B-lymphocytes    -   CD38: underexpressed on CLL cells as compared to B-cell        precursors

Example 3. Antibody Panels and Diagnostic Method for MRD Detection inMM/PCD Patients Markers for Identification of Total Plasma Cells in theBone Marrow:

List of identification markers: CD38, CD138 and CD229

How to use them: Any combination of the three markers in anyfluorochrome position works; also it is possible to use any combinationsof two of the three markers or in a subset of cases (not all) even oneof the three markers alone. Preferable combinations are order asfollows: 1) CD138/CD38/CD229; 2) CD138/CD38, 3) CD138/CD229; 4)CD38/CD229; 5) CD138; 6) CD38); 7) CD229. Note that any of these markersindividually and in combination may be used in combination also withsideward light scatter (SSC) or forward light scatter (FSC) or both FSCand SSC to identify plasma cells in the bone marrow or other types ofsamples (e.g. peripheral blood, tissue biopsy, spinal fluid).

Markers for Distinguishing Normal Vs Clonal/Malignant Plasma Cells:

List of markers and most frequent phenotypic aberration:

-   -   CD38: underexpressed in malignant plasma cells compared to        normal plasma cells    -   CD27: underexpressed in malignant plasma cells compared to        normal plasma cells    -   CD45: underexpressed in malignant plasma cells compared to        normal plasma cells    -   CD19: underexpressed (usually negative) in malignant plasma        cells compared to normal plasma cells    -   CD81: underexpressed in malignant plasma cells compared to        normal plasma cells    -   CD56: overexpressed in malignant plasma cells compared to normal        plasma cells    -   CD28: overexpressed in malignant plasma cells compared to normal        plasma cells    -   CD117: overexpressed in malignant plasma cells compared to        normal plasma cells    -   CyIgk and CyIglambda: expression restricted to either one or the        other Ig light chains in malignant plasma cells while showing a        balanced distribution    -   (CyIgk/CyIglambda ratio in normal plasma cells usually ranging        between ratios 3 and 0.5).    -   SSC: increased or decreased intensity in malignant plasma cells        compared to normal plasma cells.    -   FSC: increased or decreased intensity in malignant plasma cells        compared to normal plasma cells.

1. A reagent composition for flow cytometric detection of B-cellprecursor ALL (BCP-ALL) in a human subject, comprising a panel of atleast eight distinct fluorochrome-conjugated antibodies, the panelcomprising at least antibodies against the core markers CD10, CD19,CD20, CD34 and CD45, and wherein the panel further comprises one or moreantibodies selected from the group consisting of antibodies againstCD38, CD81, CyIgμ, and deoxynucleotidyl transferase (NuTdT), and whereinthe panel further comprises one or more sets of antibodies selected fromthe group consisting of: (a) set of antibodies against CD66c and CD123;(b) set of antibodies against CD304 and CD73; and (c) set of antibodiesagainst SmIgκ and SmIgλ, wherein the antibodies within each set areconjugated to the same fluorochrome and wherein between different setsthe fluorochromes are distinguishable.
 2. (canceled)
 3. The reagentcomposition according to claim 1, wherein the panel comprises two ormore sets of antibodies selected from the group consisting of: (a) setof antibodies against CD66c and CD123; (b) set of antibodies againstCD304 and CD73; and (c) set of antibodies against SmIgκ and SmIgλ. 4.(canceled)
 5. The reagent composition according to claim 1, comprisingdistinct fluorochrome-conjugated antibodies directed against one of thefollowing combinations of markers: (i) CD20, CD45, CD81, CD66c, CD123,CD34, CD19, CD10 and CD38, wherein the antibodies against CD66c andCD123 are conjugated to the same fluorochrome; (ii) CD20, CD45, CD81,CD304, CD73, CD34, CD19, CD10 and CD38, wherein the antibodies againstCD304 and CD73 are conjugated to the same fluorochrome; or (iii) CD20,CD45, NuTdT, SmIgκ, SmIgλ, CyIgμ, CD19, CD34 and CD10, wherein theantibodies against SmIgκ and SmIgλ are conjugated to the samefluorochrome.
 6. The reagent composition according to claim 1,comprising distinct fluorochrome-conjugated antibodies directed againstthe markers CD20, CD45, CD81, NuTdT, CD34, CD19, CD10 and CD38, and oneor more sets of antibodies selected from the group consisting of: (a)set of antibodies against CD66c and CD123; (b) set of antibodies againstCD304 and CD73; and (c) set of antibodies against SmIgκ and SmIgλ,wherein the antibodies within each set are conjugated to the samefluorochrome.
 7. The reagent composition according to claim 6,comprising distinct fluorochrome-conjugated antibodies directed againstthe markers CD20, CD45, CD81, NuTdT, CD66c, CD123, CD304, CD73, SmIgκ,SmIgλ, CyIgμ, CD34, CD19, CD10 and CD38, wherein the antibodies againsteach of the sets CD66c/CD123, CD304/CD73 and SmIgκ/SmIgλ are conjugatedto the same fluorochrome. 8-13. (canceled)
 14. A diagnostic kit for flowcytometric detection of B-cell precursor ALL (BCP-ALL), comprising atleast one reagent composition according to claim 1, optionally togetherwith instructions for use, buffer, and/or control samples.
 15. Amulti-color flow cytometric method for detecting B-cell precursor ALL(BCP-ALL) in a biological sample comprising cells, preferablylymphocytes, comprising the steps of: (i) staining the sample with areagent composition according to claim 1, (ii) subjecting the sample toflow cytometry; (iii) gating on cells for expression of the selectedmarkers detected by the antibodies present in the reagent composition;and (iv) distinguishing between normal and malignant cells, based on theexpression profile of the multiple markers.
 16. The method according toclaim 15, wherein step (iv) involves multivariate analysis, preferablyprincipal component analysis (PCA).
 17. The reagent compositionaccording to claim 1, wherein the antibody against CD20 is conjugated toPacB; the antibody against CD45 is conjugated to PacO; the antibodyagainst CD81 is conjugated to FITC; the antibody against NuTdT isconjugated to FITC; the antibody against CD66c and the antibody againstCD123 are conjugated to PE; the antibody against CD304 and the antibodyagainst CD73 are conjugated to PE; the antibody against SmIgκ and theantibody against SmIgλ are conjugated to PE; the antibody against CD34is conjugated to PerCPCy5.5; the antibody against CyIgμ is conjugated toPerCPCy5.5; the antibody against CD19 is conjugated to PECy7; theantibody against CD10 is conjugated to APC or APCC750; and the antibodyagainst CD38 is conjugated to APCC750.
 18. The diagnostic kit accordingto claim 14, wherein the antibody against CD20 is conjugated to PacB;the antibody against CD45 is conjugated to PacO; the antibody againstCD81 is conjugated to FITC; the antibody against NuTdT is conjugated toFITC; the antibody against CD66c and the antibody against CD123 areconjugated to PE; the antibody against CD304 and the antibody againstCD73 are conjugated to PE; the antibody against SmIgκ and the antibodyagainst SmIgλ are conjugated to PE; the antibody against CD34 isconjugated to PerCPCy5.5; the antibody against CyIgμ is conjugated toPerCPCy5.5; the antibody against CD19 is conjugated to PECy7; theantibody against CD10 is conjugated to APC or APCC750; and the antibodyagainst CD38 is conjugated to APCC750.
 19. The multi-color flowcytometric method according to claim 15, wherein the antibody againstCD20 is conjugated to PacB; the antibody against CD45 is conjugated toPacO; the antibody against CD81 is conjugated to FITC; the antibodyagainst NuTdT is conjugated to FITC; the antibody against CD66c and theantibody against CD123 are conjugated to PE; the antibody against CD304and the antibody against CD73 are conjugated to PE; the antibody againstSmIgκ and the antibody against SmIgλ are conjugated to PE; the antibodyagainst CD34 is conjugated to PerCPCy5.5; the antibody against CyIgμ isconjugated to PerCPCy5.5; the antibody against CD19 is conjugated toPECy7; the antibody against CD10 is conjugated to APC or APCC750; andthe antibody against CD38 is conjugated to APCC750.